The CD95 molecule, also called Fas and Apo-1, is a cell-surface receptor found on a variety of cell types including activated T and B cells. Ligation of this molecule with specific antibodies or its natural ligand (FasL) usually induces rapid and dramatic apoptotic cell death. Naturally occurring mutations in Fas or FasL in mice result in profound lymphoaccumulative diseases marked by accelerated autoimmunity. Therefore, the apoptotic signal delivered by Fas/FasL interactions in vivo are believed to be critical for normal immunologic homeostasis. Despite the importance of this interaction and the general progress made in the field of apoptosis, very little is known about the nature of the apoptotic signal delivered by Fas ligation. We and other laboratories have found that ligation of Fas induces the hydrolysis of sphingomyelin to produce ceramide, and as ceramide is capable of inducing apoptosis, this suggests that the sphingomyelin pathway is an important mediator of Fas-induced apoptosis. In this proposal, we will examine the mechanisms whereby Fas- ligation induces acidic sphingomyelinase, which in turn hydrolyzes sphingomyelin. In preliminary experiments, we have examined a Fas+ cell line from an individual with genetic defects in acidic sphingomyelinase (Niemann-Pick disease) and have found this line to be strikingly resistant to Fas- but not ceramide-induced apoptosis. Introduction of wild-type acid sphingomyelinase by retroviral gene transfer appeared to restore the Fas pathway to apoptosis. Thus, it is likely that the activation of acidic sphingomyelinase to generate ceramide represents a critical step in Fas-induced cell death. Our studies will therefore focus considerable attention on the mechanisms of activation of this enzyme and its regulation following ligation of Fas and other TNFR family receptors. In addition, we will extend this signaling pathway from ceramide to apoptosis, through the use of novel cell-free systems. In these systems, nuclei are induced to undergo morphologic changes associated with apoptosis upon addition of ceramide. These effects are dependent upon the presence of cellular extracts prepared from Xenopus eggs or a human T lymphoma line. Using these systems, we will extend our dissection of Fas- induced signals from ceramide to downstream apoptotic events. Thus, we propose a step-by-step analysis of the induction of apoptosis from Fas to sphingomyelin hydrolysis and ceramide generation to ceramide-induced signals leading to apoptosis.